【作者】 李红艳；

【导师】 尹建元；

【作者基本信息】 吉林大学 ， 药物分析学， 2022， 博士

【摘要】 牙周炎是发生在牙齿支持组织的慢性炎症性疾病,以牙龈出血、牙周袋的形成、牙槽骨的吸收为主要临床特征,是成年人牙齿缺失的首要病因,严重危害口腔健康。此外,牙周炎还与全身多种系统性疾病密切相关,影响全身健康[1]。牙周炎治疗的目标是修复牙周缺损,实现牙周组织再生。然而,牙周炎条件下各种细胞的修复、再生能力有限,牙周组织再生目前仍然是一项临床挑战。调节炎症反应,提高炎症微环境下干细胞的再生潜能对于牙周组织再生至关重要。因此,寻找、开发能够调节炎症反应,提高干细胞分化能力,促进牙周组织再生的分子或药物是目前牙周炎临床治疗的热点。研究发现,腺苷酸活化蛋白激酶(adenosine monophosphate activated protein kinase,AMPK)信号通路在炎症和骨组织代谢过程中均发挥重要的调控作用,通过AMPK信号通路可以调控巨噬细胞介导的炎症反应,促进间充质干细胞的成骨分化,促进牙周组织再生,AMPK是牙周炎治疗的有效靶点。贝派地酸(ETC-1002)是一种AMPK的激动剂,目前用于高胆固醇血症的治疗,鉴于老药新用比开发新药投入少、周期短的优点,本研究拟以AMPK作为牙周炎治疗靶点,将ETC-1002首次用于牙周炎的治疗,并探讨其中的机制。由此设计了以下3个方面的研究工作:(1)ETC-1002对牙龈卟啉单胞菌脂多糖(Porphyromonas gingivalis lipopolysaccharide,Pg-LPS)诱导的RAW264.7细胞炎症反应的影响及相关机制研究。首先通过Real-time PCR和ELISA检测ETC-1002对Pg-LPS诱导的巨噬细胞炎症因子如白介素-6(Interleukin-6,IL-6)、白介素-1β(Interleukin-1β,IL-1β)和肿瘤坏死因子-α(Tumor Necrosis Factorα,TNF-α)表达的影响,结果表明ETC-1002抑制Pg-LPS诱导的巨噬细胞炎症因子的表达,抑制巨噬细胞炎症反应。然后,通过Western blot检测了ETC-1002在炎症和正常状态下对AMPKα磷酸化的影响,结果表明ETC-1002在炎症和正常状态下均促进AMPKα的磷酸化。接下来,为了验证AMPK信号通路在ETC-1002抑制巨噬细胞炎症反应中的作用,阻断AMPK信号通路,应用Real-time PCR和Western blot检测巨噬细胞中炎症因子的表达,结果表明,在AMPK信号通路被阻断后,巨噬细胞炎症因子的表达明显升高,充分证明ETC-1002通过激活AMPK信号通路抑制巨噬细胞的炎症反应。NF-κB信号通路是牙周炎症反应的经典通路,为了阐明AMPK和NF-κB两个信号通路在ETC-1002抑制巨噬细胞炎症中的具体作用关系,我们应用Western blot检测了炎症状态下、ETC-1002作用后及阻断AMPK通路后,NF-κB信号通路相关蛋白IκBα和p65的磷酸化水平。结果发现,Pg-LPS提高了IκBα和p65的磷酸化,激活了NF-κB信号通路;ETC-1002降低了IκBα和p65的磷酸化水平,抑制了炎症状态下NF-κB信号通路的激活;而阻断AMPK通路后,IκBα和p65的磷酸化水平再次升高,ETC-1002对NF-κB信号通路的抑制作用消失。这些研究结果表明ETC-1002促进RAW264.7细胞AMPKα的磷酸化激活,从而负性调节NF-κB信号通路,抑制炎性细胞因子的产生,从而发挥抗炎作用。(2)在炎症及正常状态下ETC-1002对大鼠骨髓间充质干细胞(rat bone marrow mesenchymal stem cells,r BMSCs)成骨分化的影响及相关机制研究。首先,通过Real-time PCR检测不同浓度ETC-1002作用下,成骨基因Runt相关转录因2(Runt-related transcription factor 2,RUNX2)、Osterix(OSX)、碱性磷酸酶(alkaline phosphatase,ALP)和I型胶原(collagen type I,COL1)的表达变化,确定ETC-1002促进r BMSCs成骨分化的有效作用浓度,结果表明,100μM的ETC-1002作用最强。然后,我们通过Real-time PCR检测早期成骨基因的表达,进一步确定在炎症及正常状态下ETC-1002对r BMSCs成骨分化的影响,结果发现,ETC-1002在炎症及正常状态下均可以促进成骨基因的表达。然后,通过ALP染色检测了ALP的表达及活性,表明在炎症及正常状态下ETC-1002均可以提高r BMSCs中ALP的表达和活性。接下来,为了评估ETC-1002的晚期成骨分化能力,我们通过茜素红染色(Alizarin red staining,ARS)检测钙结节的形成,结果表明,在炎症及正常状态下ETC-1002均可以促进r BMSCs中钙结节的形成。最后,我们对ETC-1002促进r BMSCs成骨分化的机制进行了相关研究。分别在炎症及正常状态下,应用AMPK的抑制剂Compound C阻断r BMSCs中的AMPK信号通路,进行Real-time PCR、ALP染色和ARS染色以评估ETC-1002诱导的成骨分化是否受到影响。结果表明,无论在炎症还是在正常状态下,阻断AMPK信号通路后,ETC-1002诱导的r BMSCs成骨因子的表达、ALP的表达及钙结节的形成均受到明显抑制。这充分证明了,无论在炎症还是正常状态下AMPK信号通路均参与了ETC-1002诱导的r BMSCs的成骨分化过程。因此,我们通过这部分实验证明了,无论在炎症还是正常状态下,ETC-1002均可通过激活AMPK信号通路促进r BMSCs的成骨分化。(3)ETC-1002对小鼠实验性牙周炎的作用及相关机制研究。应用C57BL/6小鼠建立实验性牙周炎模型,将其分为牙周炎组、ETC-1002组和Compound C组。十天后,通过检测牙周临床指标发现ETC-1002减小龈沟出血指数和牙齿松动指数,改善小鼠牙周炎症状。为了明确ETC-1002对牙周炎症的影响,我们通过HE染色,观察上颌第一、二磨牙之间牙周组织结构的改变和炎症细胞的数量和分布。结果表明,牙周炎组牙周组织内可见大量炎性细胞浸润,结缔组织附着丧失,牙周纤维排列紊乱,牙槽骨吸收明显;ETC-1002组中少量炎性细胞散在分布,胶原纤维排列较为规则;Compound C阻断AMPK通路后牙周组织再次出现大量淋巴细胞和浆细胞浸润。证明ETC-1002通过AMPK通路发挥抗炎作用。为了进一步验证ETC-1002的抗炎作用,应用Real-time PCR和免疫组化检测牙周组织炎症胞因子(IL-6、IL-1β和TNF-α)的表达。结果发现,牙周炎组炎症因子表达增高;ETC-1002组炎症因子表达降低;阻断AMPK通路后,炎症因子的表达再次升高。再次证明了ETC-1002通过AMPK通路发挥抗炎作用。为了明确ETC-1002的体内促成骨作用,对上颌右侧第一、二磨牙进行micro-CT扫描,测量并分析釉牙骨质界(CEJ)到牙槽嵴顶(ABC)之间的距离、相关骨及骨小梁参数。结果发现,牙周炎组牙槽骨吸收严重,骨量明显减少,骨矿化密度降低;ETC-1002组牙槽骨的高度、骨量和骨矿化密度均明显增加;阻断AMPK通路后,牙槽骨的高度、骨量和骨矿化密度均再次显著降低。由此说明ETC-1002通过AMPK通路促进牙槽骨再生。为了进一步验证ETC-1002的促成骨分化作用,应用Real-time PCR检测牙周组织成骨相关因子(RUNX2、OSX、COL1、ALP、OCN和OPN)的表达。结果发现牙周炎组成骨因子表达降低;ETC-1002组成骨因子表达增高;阻断AMPK后,成骨因子表达再次降低。再次证明ETC-1002通过AMPK通路发挥促成骨作用。此外,为了观察ETC-1002的体内生物安全性,我们对小鼠的肝、肾、脾和胸腺的组织切片进行HE染色,并检测血液中谷草转氨酶(aspartate aminotransferase,AST)、谷丙转氨酶(alanine aminotransferase,ALT)和肌酐的含量。结果显示,与对照组相比,ETC-1002组的细胞形态及血液中AST、ALT和肌酐的含量均未见明显异常,说明ETC-1002体内生物安全性较好。因此,这部分实验证明了ETC-1002在体内发挥抗炎和促成骨作用,并且具有良好的生物安全性。综上,本研究证实ETC-1002可以通过AMPK/NF-κB通路抑制Pg-LPS诱导的巨噬细胞炎症反应,通过AMPK通路促进炎症及正常状态下r BMSCs的成骨分化。体内实验结果表明,ETC-1002通过AMPK通路抑制小鼠牙周炎症,促进牙槽骨修复、再生。由此可见,ETC-1002通过AMPK在牙周炎中发挥治疗作用,可以作为牙周炎治疗的候选药物,为牙周炎的临床治疗提供新的思路。更多还原

【Abstract】 Periodontitis is a chronic inflammatory disease characterized by bleeding gums,periodontal pocket formation and alveolar bone resorption,which ultimately leads to tooth loss.Periodontitis not only harms the oral function,but is also closely related to systemic diseases.The goal of periodontitis treatment is to achieve periodontal tissue regeneration.At present,none of the traditional treatment methods in the clinic can obtain complete periodontal regeneration.Periodontal regeneration efforts have shown limited success due to the inability to control the inflammation that negatively affects stem cell function.Resolution of inflammation may be a possible key early event in periodontal regeneration,by kickstarting the resolution of the inflammation process and creating a pro-regenerative environment for periodontal tissue regeneration that is otherwise inhibited by local inflammation.Strategies other than resolution of inflammation that could promote stem cell regeneration are also important.Therefore,it is urgent to develop effective treatments which is able to both reduce the infections and inflammation in injured sites and improve the regeneration of damaged tissues.AMP-activated protein kinase(AMPK)plays a regulatory role in inflammation and bone metabolic processes.Bempedoic acid(ETC-1002),a pharmacological activator of AMPK,showed a promising beneficial effect in in vivo models of inflammation,indicating this drug as an alternative tool to manage periodontitis.Given that,we visualize that ETC-1002 may promote periodontal tissue regeneration by modulating macrophage mediated inflammatory responses and promoting the osteogenic differentiation of r BMSCs,through AMPK pathway.Subsequently,specific research work was elaborated from the following three aspects:(1)The role and mechanism of ETC-1002 on inflammation in RAW264.7 induced by Pg-LPS.First,we examined the effects of ETC-1002 on the expression of inflammatory cytokines(IL-6,IL-1β and TNF-α)in macrophage by Real-time PCR and ELISA.The results suggest that ETC-1002 inhibits expression of inflammatory cytokines and alleviates inflammatory responses in macrophage.Then,the effect of ETC-1002 on phosphorylation of AMPK under inflammatory and normal conditions was examined by Western blot.The results indicate that ETC-1002 promotes phosphorylation of AMPK under both inflammatory and normal conditions.Next,to verify the role of AMPK signaling pathway in ETC-1002-inhibited inflammatory responses in macrophages,AMPKαsi RNA was applied to block the AMPK signaling pathway,and the expression of inflammatory cytokines were detected in macrophages by Real-time PCR and Western blot.The results show that the expression of inflammatory cytokines in macrophages was significantly decreased after the AMPK signaling pathway blocked,suggesting that ETC-1002 inhibits inflammatory responses in macrophages through AMPK signaling pathway.NF-κB signaling pathway is a classical signaling pathway in periodontal inflammatory response.In order to clarify the relationship between AMPK and NF-κB signaling pathways in the process of inflammation inhibited by ETC-1002,the phosphorylation levels of IκBα and p65 were examined by Western blot,when Pg-LPS,ETC-1002 or AMPK inhibitors were added.The results show that Pg-LPS promotes the phosphorylation of IκBα and p65,and activates NF-κB signaling pathway;ETC-1002 reduces the phosphorylation of IκBαand p65,and suppresses NF-κB signaling pathways in inflammatory conditions;however,the phosphorylation of IκBα and p65 is elevated again after blocking the AMPK signaling pathway,and the inhibition of NF-κB signaling pathway by ETC-1002 is disappeared.These results suggest that ETC-1002 exerts anti-inflammatory effects by promoting AMPKα phosphorylation and negatively regulating NF-κB signaling pathway.(2)Effects and mechanisms of osteogenic differentiation in r BMSCs promoted by ETC-1002 under inflammatory and normal conditions.First,the expression of osteogenic genes(RUNX2,OSX,ALP and COL1)by Real-time PCR were performed to determine the effective concentration of ETC-1002 on the osteogenic differentiation of r BMSCs.The results show that 100 μM is the most effective concentration of ETC-1002.Then,in order to determine the effects of ETC-1002 on the osteogenic differentiation of r BMSCs under inflammatory and normal conditions,the Real-time PCR was performed to detect the expression of early osteogenic differentiation genes(RUNX2,OSX,ALP and COL1).It is found that ETC-1002 can promote gene expression of RUNX2,OSX,ALP and COL1 in r BMSCs under both inflammatory and normal conditions.In addition,the expression of ALP was detected by ALP staining.The results indicate that ETC-1002 can promote the expression of ALP in r BMSCs under inflammatory and normal conditions.We further detected the formation of calcium nodules by ARS staining and evaluated the ability of r BMSCs osteogenic differentiation in later stage.The results show that ETC-1002 can promote the formation of calcium nodules in r BMSCs under inflammatory and normal conditions.Finally,we explored the mechanism of ETC-1002 promoting osteogenic differentiation of r BMSCs under inflammatory and normal conditions.The AMPK inhibitor Compound C was used to block AMPK signaling pathway,and Real-time PCR,ALP staining,and ARS were performed to evaluate the effects of ETC-1002 on the osteogenic differentiation of r BMSCs under inflammatory and normal conditions.The results show that the expression of osteogenic differentiation genes and ALP and formation of calcium nodule in r BMSCs induced by ETC-1002 were inhibited after AMPK signaling pathway blocked,under inflammatory and normal conditions.It is well documented that AMPK signaling pathway is involved in the process of ETC-1002-induced osteogenic differentiation of r BMSCs under inflammation and normal conditions.Therefore,in this part,it is found that ETC-1002 can promote the osteogenic differentiation of r BMSCs by through AMPK signaling pathway both under inflammation and normal conditions.(3)The effects of ETC-1002 on experimental periodontitis in mice were evaluated.C57 BL / 6 mice were used to establish the periodontitis model,which were divided into three groups: Periodontitis group,ETC-1002 group and Compound C group.After 10 days,it is found that ETC-1002 reduces sulcus bleeding index and tooth mobility index and improves periodontitis symptoms in mice.The samples were scanned by micro-CT and the distance from the enamel cementum boundary(CEJ)to the top of the alveolar ridge(ABC)in the maxillary first molars were measured as the index of alveolar bone loss.The relevant bone and bone trabecular parameters were also measured between the maxillary first and second molars.It is found that the alveolar bone resorption is severe in the periodontitis group.Moreover,the bone mass and bone mineralization density are also decreased.To our delight,the height of alveolar bone,bone mass,and bone mineralization density are almost restored to the normal level in the ETC-1002 group.But,the alveolar bone resorption is severe,and the bone mass and bone mineralization density are also decreased again,after the AMPK signaling pathway inhibited.We illustrate that ETC-1002 promotes alveolar bone regeneration through the AMPK signaling pathway.To further verify the effect of ETC-1002 on the osteogenic differentiation,the expression of osteogenesis genes(RUNX2,OSX,COL1,ALP,OCN and OPN)in periodontium was examined by Real-time PCR.The results show that the expression of osteogenesis genes(RUNX2,OSX,COL1,ALP,OCN and OPN)were decreased in the periodontitis group,and increased in the ETC-1002 group.But,the expression of osteogenesis genes was decreased again,after AMPK signaling pathway inhibited.We demonstrate that ETC-1002 exerts promoting osteogenic effects through AMPK signaling pathway.To clarify the effect of ETC-1002 on inflammation of periodontium,HE staining was performed to observe the distribution of inflammatory cells in the periodontium between the first and second maxillary molars.The results show that a large number of inflammatory cells are observed in the periodontium of the periodontitis group,with loss of connective tissue attachment,disarrangement of periodontal fibers and resorption of alveolar bone.Scattered inflammatory cells and regular arrangement of collagen fibers are observed in the ETC-1002 group.But,massive lymphocyte and plasma cells infiltrate again in the periodontium,after AMPK signaling pathway inhibited.We demonstrate that ETC-1002 exerts anti-inflammatory effects through AMPK signaling pathways.To verify the anti-inflammatory effect of ETC-1002 furtherly,the expression of inflammatory cytokines(IL-6,IL-1 β and TNF-α)was detected by Real-time PCR.It is found that the expression of inflammatory cytokines was increased in the periodontitis group,descended in the ETC-1002 group,and increased again in the Compound C group.It proves again that ETC-1002 exerts anti-inflammatory effects through AMPK signaling pathway.In addition,the biosafety of ETC-1002 in vivo was evaluated by HE staining based on vital organs,including thymus,livers,spleens,and kidneys,as well as detecting the contents of AST,ALT and creatinine in blood.The results show that here is no significant difference in the cell morphology and contents of AST,ALT and Creatinine in blood between the control and the ETC-1002 group.Therefore,this part of the experiment demonstrates that ETC-1002 exerts anti-inflammatory and promoting osteogenic effects in vivo,and are safe for applications.In summary,we demonstrate that ETC-1002 alleviates inflammation in macrophages induced by Pg-LPS through AMPK/NF-κB signaling pathway and promotes osteogenic differentiation of r BMSCs through AMPK signaling pathway under both inflammatory and normal conditions.Importantly,ETC-1002 can inhibit inflammation of periodontium and enhance alveolar bone regeneration through AMPK pathway in mice with periodontitis vivo.It follows that ETC-1002 plays a therapeutic role in periodontitis via AMPK signaling pathway and it may serve as a candidate drug for periodontitis treatment.更多还原